Phage display technology - Applications and …

1 Phage display technology - Applications and innovationsMarco Antonio ArapUniversidade de S o Paulo, Hospital das Clinicas da Faculdade de Medicina,Departamento de Cirurgia, Disciplina de Urologia, S o Paulo, SP, expression of exogenous peptides on the surface of filamentous bacteriophage was initially described by Smithin 1985. Since his first study, different molecules such as small peptides and antibodies have been displayed on coatproteins of Phage , greatly expanding the Applications of the technology . The past decade has seen considerableprogress in the techniques and Applications of Phage libraries. In addition, different screening methods have allowedisolation and characterization of peptides binding to several molecules in vitro, in the context of living cells, in animalsand in humans.

2 Here we review the Applications , recent innovations, and future directions of Phage words: Phage display , : March 15, 2004; Accepted: October 19, display technology was first introduced in1985 by George Smith . It was used as an expression vector,capable of presenting a foreign amino acid sequence acces-sible to binding an antibody. Since then, a large number ofphage displayed peptide and protein libraries have beenconstructed (Basset , McCaffertyet ,Barbaset , Smith 1991, Smith and Scott 1993,Hoogenboom 2002, Szardenings 2003), leading to varioustechniques for screening such libraries. This technologyhas had a major influence on the work and discoveries donein the fields of immunology, cell biology, pharmacologyand drug display allows the presentation of large peptideand protein libraries on the surface of filamentous Phage ,which leads to the selection of peptides and proteins, in-cluding antibodies, with high affinity and specificity to al-most any target.

3 The technology involves the introductionof exogenous peptide sequences into a location in the ge-nome of the Phage capsid proteins. The encoded peptidesare expressed or displayed on the Phage surface as a fu-sion product with one of the Phage coat proteins. This way,instead of having to genetically engineer different proteinsor peptides one at a time and then express, purify, and ana-lyze each variant, Phage display libraries containing up to1010variants can be constructed simultaneously. Phage par-ticles withstand very harsh conditions, such as low pH andlow temperatures, without losing bacterial , protocols using low pH and high concentration ureahave been used to dissociate bound Phage from a target.

4 Inaddition, bound Phage does not need to be eluted from amicrotiter well or animal tissue before bacterial , infection can proceed after addition of bacteria di-rectly into the well or to the homogenized organ or strength of Phage technology is its ability to iden-tify interactive regions of proteins and other moleculeswithout preexisting notions about the nature of the interac-tion. The past decade has seen considerable progress in theapplications of Phage display technology . Different screen-ing methods have allowed isolation and characterization ofpeptides binding to several molecules in vitro, in the con-text of living cells, in animals and in humans (Arap 2002b).

5 Here we review the Applications , as well as recent innova-tions and future directions of Phage display - Structure and biologyThe bacteriophage (or simply Phage ) mostly used inphage display technology , are single-stranded DNA virusesthat infect a number of gram-negative bacteria. The fila-mentous Phage particles mostly used for display purposesare known as Ff and include strains M13, f1, Fd and ft. Fdphage particles consist of a long cylindrical protein capsid,930 nm in length and nm in diameter, enclosing a sin-gle-stranded DNA genome of about 6400 nucleotides, con-sisting of 11 genes. The viral mass is approximatelyGenetics and Molecular Biology, 28, 1, 1-9 (2005)Copyright by the Brazilian Society of Genetics.

6 Printed in correspondence to Marco Antonio Arap. Universidade de S oPaulo, Hospital das Clinicas da Faculdade de Medicina, Depar-tamento de Cirurgia, Disciplina de Urologia, R. Adma Jafet 50, 3oandar, Bela Vista 01308-050 S o Paulo, SP, Brazil. MDa, and consists mainly of about 2700 copies of thepVIII, a 50 aa residue protein encoded bygene of the Phage particle there are 3 to 5 copies of the pro-teins pVII and pXIX (genes VII and XIX) and on the otherside there are 3 to 5 copies of the proteins pIII and pVI (Fig-ure 1) (Webster 2001). In most display Applications , pIII, a406 aa adsorption protein, is the protein used for peptide ex-pression.

7 The pIII protein appears to have two functionaldomains: an exposed N-terminal domain that binds the Fpilus, but is not required for Phage particle assembly, and aC-terminal domain that is buried in the particle and is an in-tegral part of the capsid structure. The C-terminal portion ofpVIII is inside the Phage particle, close to the DNA, whilethe N-terminal part is exposed to the particles are able to infect a variety of Gram-negative bacteria, includingE. coli,using pili (F pilus ) as receptors. Filamentous Phage infection does notproduce lytic infection inE coli., but rather induces a statein which the infected bacteria produce and secrete phageparticles into the growing medium.

8 Infection begins by theattachment of Phage pIII to the F pilus of a maleE. coli. Thecircular single-stranded DNA enters the bacteria where it isconverted by the host DNA replication machinery into dou-ble-stranded plasmid replicative form. By rolling circlereplication, the replicative form makes single-strandedDNA and the templates for expression of proteins pIII andpVIII are formed. Phage descendants are assembled bypackaging of the single-stranded DNA into protein coatsand extruded through the bacterial membrane (Russel1991).Most of the currently used Phage display vectors usethe N-terminus of pIII protein or pVIII protein to displaythe foreign peptide or protein (Smith and Scott 1993).

9 ThepIII libraries display 3-5 copies of each individual peptide(Scott and Smith 1990), whereas pVIII libraries can displayup to 2700 copies of small (up to six amino acids) peptides(Greenwoodet ). The pIII and pVIII proteins candisplay peptides of various lengths and cysteine residuescan be introduced to the fusion peptide to createconformational constraints by the formation of loops be-tween disulfide bridged cysteine residues. Furthermore, theexogenous peptides are well exposed, facilitating the in-sert-target interactions. Large peptide inserts of up to 38amino acids can be introduced into the amino terminus ofpIII protein without the loss of Phage infectivity or with Phage display technologyDetailed description of the materials, methods andspace needed for those who want to start Phage work maybe found in specialized textbooks (Barbaset ,Pasqualini and Arap 2002).

10 The basic protocols used withphage studies can also be obtained in these books, whereasits variations are found in published studies. Initial invest-ment is relatively small, as most of the fundamental materi-als are common laboratory devices, such as Petri dishes,Falcon tubes and centrifuges. However, one cannot forgetthe most important tool for Phage work: k91kan E. coli andpeptide or antibody Phage libraries. The construction of apeptide Phage library involves a detailed protocol and, forthose who are not experienced with Phage work, one way toobtain reliable Phage libraries and start a bio-panning isto establish a collaboration with a more experienced labora-tory.